A four-wavelength microspectrophotometric method has been developed to determine both O2 and CO saturation of hemoglobin in frozen small vessels. This method compensates for the nonspecific light loss related to the ice crystals and in addition can simultaneously measure both hemoglobin O2 and CO saturation in frozen blood. By using four wavelengths in the 540-580 nm range, it is possible to determine both O2 and CO saturations in frozen blood. This four-wavelength method demonstrated an accuracy of 6-8% within 95% confidence limits (SE of estimate = 3-4%) for both CO and O2 saturation determinations in frozen blood. The linear regression of the hemoglobin O2 saturation calibration was significant with an r2 value of 0.989. The linear regression of the CO saturation calibration was also significant with an r2 of 0.996. The repeatability of each measurement was on the order of 1-6%. This method also represents an accuracy within 5% for measurements in the frozen rabbit heart for both CO and O2 in vessels between 20 and 100 microns in diameter. It compares favorably with other previously reported methods. It shows a similar accuracy but requires fewer repeat measurements than the three-wavelength method. This method can provide accurate measurement of O2 and CO saturation in small frozen arterioles and venules of any organ.